Skip to main content
SpringerLink
Log in

Imaging the accumulated intracellular microalgal lipids as a response to temperature stress

  • Original Article
  • Published:
3 Biotech Aims and scope Submit manuscript

Abstract

Over the last few decades, many scientists considered microalgae as promising actors for future biofuels because of the high lipid productivity inside their cells. Moreover, much attention has been paid to algal lipids as they can be used in biodiesel production. In this study, we optimized the different suitable conditions such as incubation time, incubation temperature, Dimethylesulfoxide and Nile red concentrations of the lipophilic fluorescence dye Nile red as an excellent and fast vital stain to detect and quantify intracellular lipids. This was achieved using the green alga Nannochloropsis salina. In addition, investigating the accumulation of lipid vesicles inside different isolated microalgal species as a response to temperature stress. Furthermore, the confocal laser scanning microscopy (LS510) for imaging and measuring the size and volume of the accumulated lipid vesicles was used.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  • Abou-Shanab RAI, Matter IA, Kim SN, Oh YK, ChoiI Jeon BH (2011) Characterization and identification of lipid-producing microalgae species isolated from a freshwater lake. Biomass Bioenerg 35(7):3079–3085. doi:10.1016/j.biombioe.2011.04.021

    Article  CAS  Google Scholar 

  • Akimoto S, Mimuro M (2007) Application of time-resolved polarization fluorescence spectroscopy in the femtosecond range to photosynthetic systems. Photochem Photobiol 83:163–170. doi:10.1562/2006-02-28-IR-825

    CAS  Google Scholar 

  • Bertozzini E, Galluzzi L, Penna A, Magnani M (2011) Applications of the standard addition method for the absolute quantification of neutral lipids in microalgae using Nile red. J Microbiol Meth 87(1):17–23. doi:10.1016/j.mimet.2011.06.018

    Article  CAS  Google Scholar 

  • Brown WJ, Sullivan TR, Greenspan P (1992) Nile red staining of lysosomal phospholipid inclusions. Histochemistry 97(4):349–354

    Article  CAS  Google Scholar 

  • Cabanelas IT, Van der Zwart M, Kleinegris DM, Barbosa MJ, Wijffels RH (2015) Rapid method to screen and sort lipid accumulating microalgae. Bioresour Technol 18:47–52. doi:10.1016/j.biortech.2014.10.057

    Article  Google Scholar 

  • Chen W, Zhang C, Song L, Sommerfeld M, Hu Q (2009) A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae. J Microbiol Meth 77(1):41–47. doi:10.1016/j.mimet.2009.01.001

    Article  CAS  Google Scholar 

  • Chen M, Tang H, Ma H, Holland TC, Ng KYS, Salley SO (2011) Effect of nutrients on growth and lipid accumulation in the green alga Dunaliella tertiolecta. Bioresour Technol 102(2):1649–1655. doi:10.1016/j.biortech.2010.09.062

    Article  CAS  Google Scholar 

  • Chisti Y (2007) Biodiesel from microalgae. Biotechnol Adv 25:294–306. doi:10.1016/j.biotechadv.2007.02.001

    Article  CAS  Google Scholar 

  • Cho K, Kim KN, Lim NL, Kim MS, Ha JC, Shin HH, Mi-Kyung Roh SW, Kim D, Oda T (2015) Enhanced biomass and lipid production by supplement of myo-inositol with oceanic microalga Dunaliella salina. Biomass Bioenerg 72:1–7. doi:10.1016/j.biombioe.2014.11.014

    Article  CAS  Google Scholar 

  • Cirulis JT, Strasser BC, Scott JA, Ross GM (2012) Optimization of staining conditions for microalgae with three lipophilic dyes to reduce precipitation and fluorescence variability. Cytom A 81A:618–626. doi:10.1002/cyto.a.22066

    Article  CAS  Google Scholar 

  • Collet P, Lardon L, Helias A, Bricout S, Lombaert-Valot I, Perrier B, Lepine O, Steyer JP (2014) Biodiesel from microalgae-life cycle assessment and recommendations for potential improvements. Renew Energy 71:525–533. doi:10.1016/j.renene.2014.06.009

    Article  CAS  Google Scholar 

  • Cooper MS, Hardin WR, Petersen TW, Cattolico RA (2010) Visualizing “green oil” in live algal cells. J Biosci Bioeng 109(2):198–201. doi:10.1016/j.jbiosc.2009.08.004

    Article  CAS  Google Scholar 

  • Doan TTY, Obbard JP (2012) Enhanced intracellular lipid in Nannochloropsis sp. via random mutagenesis and flow-cytometric cell sorting. Algal Res 1(1):17–21. doi:10.1016/j.algal.2012.03.001

    Article  CAS  Google Scholar 

  • Dunstan GA, Volkman JK, Barrett SM, Garland CD (1993) Changes in the lipid composition and maximization of the polyunsaturated fatty acid content of three microalgae grown in mass culture. J Appl Phycol 5(1):71–83. doi:10.1007/BF02182424

    Article  CAS  Google Scholar 

  • Eibl JK, Corcoran JD, Senhorinho GNA, Zhang K, Hosseini NS, Marsden J, Laamanen CA, Scott JA, Ross GM (2014) Bioprospecting for acidophilic lipid-rich green microalgae isolated from abandoned mine site water bodies. AMB Express. doi:10.1186/2191-0855-4-7

    Google Scholar 

  • Elsey D, Jameson D, Raleigh B, Cooney MJ (2007) Fluorescent measurement of microalgal neutral lipids. J Microbiol Methods 68:639–642. doi:10.1016/j.mimet.2006.11.008

    Article  CAS  Google Scholar 

  • Fabiano CB, Ernani SA, Mauricio VB, Jorge Luiz BO (2006) Lipids, fatty acids composition and carotenoids of Chlorella vulgaris cultivated in a hydroponic wastewater. Grasas Aceites 57(3):270–274. doi:10.3989/gya.2006.v57.i3.48

    Google Scholar 

  • Goncalves EC, Wilkie AC, Kirst M, Rathinasabapathi B (2016) Metabolic regulation of triacylglycerol accumulation in the green algae: identification of potential targets for engineering to improve oil yield. J Plant Biotechnol 14(8):1649–1660. doi:10.1111/pbi.12523

    Article  CAS  Google Scholar 

  • Gong Y, Guo X, Wan X, Liang L, Jiang M (2013) Triacylglycerol accumulation and change in fatty acids content of four marine oleaginous microalgae under nutrient limitation and at different culture ages. J Basic Microbial 53(1):29–36. doi:10.1002/jobm.201100487

    Article  CAS  Google Scholar 

  • Govender T, Ramanna L, Rawat I, Bux F (2012) BODIPY staining, an alternative to the Nile Red fluorescence method for the evaluation of intracellular lipids in microalgae. Bioresour Technol 114:507–511. doi:10.1016/j.biortech.2012.03.024

    Article  CAS  Google Scholar 

  • Greenspan P, Fowler SD (1985) Spectrofluorometric studies of the lipid probe, Nile red. J Lipid Res 26(7):781–789

    CAS  Google Scholar 

  • Gusbeth CA, Eing C, Göttel M, Sträßner R, Frey W (2016) Fluorescence diagnostic for lipid status monitoring of microalgae during cultivation. Int J Renew Energy Biofuels 2016:1–12. doi:10.5171/2016.899698

    Article  Google Scholar 

  • Harwati TU, Willke T, Vorlop KD (2012) Characterization of the lipid accumulation in a tropical freshwater microalgae Chlorococcum sp. Bioresour Technol 121:54–60. doi:10.1016/j.biortech.2012.06.098

    Article  CAS  Google Scholar 

  • Hu Q, Sommerfeld M, Jarvis E, Ghirardi M, Posewitz M, Seibert M, Darzins A (2008) Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J 54:621–639. doi:10.1111/j.1365-313X.2008.03492.x

    Article  CAS  Google Scholar 

  • Knothe G (2011) A technical evaluation of biodiesel from vegetable oils vs. algae. Will algae-derived biodiesel perform? Green Chem 13(11):3048–3065. doi:10.1039/C0GC00946F

    Article  CAS  Google Scholar 

  • Kou Z, Bei S, Sun J, Pan J (2013) Fluorescent measurement of lipid content in the model organism Chlamydomonas reinhardtii. J Appl Phycol 25(6):1633–1641. doi:10.1007/s10811-013-0011-x

    Article  CAS  Google Scholar 

  • Liu ZY, Wang GC, Zhou BC (2008) Effect of iron on growth and lipid accumulation in Chlorella vulgaris. Bioresour Technol 99(11):4717–4722. doi:10.1016/j.biortech.2007.09.073

    Article  CAS  Google Scholar 

  • Meng X, Yang J, Xu X, Zhang L, Nie Q, Xian M (2009) Biodiesel production from oleaginous microorganisms. Renew Energy 34(1):1–5. doi:10.1016/j.renene.2008.04.014

    Article  Google Scholar 

  • Miao X, Wu Q (2006) Biodiesel production from heterotrophic microalgal oil. Bioresour Technol 97:841–846. doi:10.1016/j.biortech.2005.04.008

    Article  CAS  Google Scholar 

  • Minhas AK, Hodgson P, Barrow CJ, Adholeya A (2016) A review on the assessment of stress conditions for simultaneous production of microalgal lipids and carotenoids. Front Microbial 7:546. doi:10.3389/fmicb.2016.00546

    Google Scholar 

  • Mishra SK, Suh WI, Farooq W, Moon M, Shrivastav A, Park MS, Yang JW (2014) Rapid quantification of microalgal lipids in aqueous medium by a simple colorimetric method. Bioresour Technol 155:330–333. doi:10.1016/j.biortech.2013.12.077

    Article  CAS  Google Scholar 

  • Morowvat MH, Rasoul-Amini S, Ghasemi Y (2010) Chlamydomonus as a “new” organism for biodiesel production. Bioresour Technol 101(6):2059–2062. doi:10.1016/j.biortech.2009.11.032

    Article  CAS  Google Scholar 

  • Natunen K, Seppala J, Schwenk D, Rischer H, Spilling K, Tamminen T (2015) Nile Red staining of phyotoplankton neutral lipids: species-specific fluorescence kinetics in various solvents. J Appl Phycol 27(3):1161–1168. doi:10.1007/s10811-014-0404-5

    Article  CAS  Google Scholar 

  • Ota S, Oshima K, Yamazaki T, Kim S, Yu Z, Yoshihara M, Takeda K, Takeshita T, Hirata A, Bišová K, Zachleder V, Hattori M, Kawano S (2016) Highly efficient lipid production in the green alga Parachlorella kessleri: draft genome and transcriptome endorsed by whole-cell 3D ultra-structure. Biotechnol Biofuels 9:13. doi:10.1186/s13068-016-0424-2

    Article  Google Scholar 

  • Park JW, Na SC, Lee Y, Lee S, Park SB, LiJeon N (2013) Measurement of lipid droplet accumulation kinetics in Chlamydomonus reinhardtii using seoul-fluor. Energies 6(11):5703–5716. doi:10.3390/en6115703

    Article  Google Scholar 

  • Pick U, Rachutin-Zalogin T (2012) Kinetic anomalies in the interactions of Nile red with microalgae. J Microbiol Meth 88(2):189–196. doi:10.1016/j.mimet.2011.10.008

    Article  CAS  Google Scholar 

  • Ren HY, Liu BF, Ma C, Zhao L, Ren NQ (2013) A new lipid-rich microalga Scenedesmus sp. strain R-16 isolated using Nile red staining: effects of carbon and nitrogen sources and initial pH on the biomass and lipid production. Biotechnol Biofuels 6:143. doi:10.1186/1754-6834-6-143

    Article  CAS  Google Scholar 

  • Romano I, Bellitti MR, Nicolaus B, Lama L, Manca MC, Pagnotta E, Gambacorta A (2000) Lipid profile: a useful chemotaxonomic marker for classification of a new cyanobacterium in Spirulina genus. Phytochemistry 54(3):289–294. doi:10.1016/S0031-9422(00)00090-X

    Article  CAS  Google Scholar 

  • Rumin J, Bonnefond H, Saint-Jean B, Rouxel C, Sciandra A, Bernard O, Cadoret JP, Bougaran G (2015) The use of fluorescent Nile red and BODIPY for lipid measurement in microalgae. Biotechnol Biofuels 8:42. doi:10.1186/s13068-015-0220-4

    Article  Google Scholar 

  • Sakthivel R, Elumalai S, Arif MM (2011) Microalgae lipid research, past, present: a critical review for biodiesel production, in the future. J Exp Sci 2(10):29–49

    Google Scholar 

  • Sharma KK, Schuhmann H, Schenk PM (2012) High lipid induction in microalgae for biodiesel production. Energies 5(5):1532–1553. doi:10.3390/en5051532

    Article  CAS  Google Scholar 

  • Shrivastava A, Gupta VB (2011) Methods for the determination of limit of detection and limit of quantitation of the analytical methods. Chron Young Sci 2(1):21–25. doi:10.4103/2229-5186.79345

    Article  Google Scholar 

  • Shuo Y, Anders B, Helge E, Claes G (2012) Neutral lipid accumulation at elevated temperature in conditional mutants of two microalgae species. Plant Physiol Biochem 61C:71–79. doi:10.1016/j.plaphy.2012.09.007

    Google Scholar 

  • Siaut M, Cuiné S, Cagnon C, Fessler B, Nguyen M, Carrier P, Beyly A, Beisson F, Triantaphylidès C, Li-Beisson Y, Peltier G (2011) Oil accumulation in the model green alga Chlamydomonas reinhardtii: characterization, variability between common laboratory strains and relationship with starch reserves. BMC Biotechnol 11:1–15. doi:10.1186/1472-6750-11-7

    Article  Google Scholar 

  • Song D, Fu J, Shi D (2008) Exploitation of oil-bearing microalgae for biodiesel. Chin J Biotechnol 24(3):341–348. doi:10.1016/S1872-2075(08)60016-3

    Article  CAS  Google Scholar 

  • Teo CL, Atta M, Bukhari A, Taisir M, Yusuf AM, Idris A (2014) Enhancing growth and lipid production of marine microalgae for biodiesel production via the use of different LED wavelengths. Bioresour Technol 162(2C):38–44. doi:10.1016/j.biortech.2014.03.113

    Article  CAS  Google Scholar 

  • Velmurugan N, Sung M, Yim SS, Park MS, Yang JW, Jeong KJ (2013) Evaluation of intracellular lipid bodies in Chylamydomonas reinhardtii strains by flow cytometry. Bioresour Technol 138:30–37. doi:10.1016/j.biortech.2013.03.078

    Article  CAS  Google Scholar 

  • Wahlen BD, Willis RM, Seefeldt LC (2011) Biodiesel production by simultaneous extraction and conversion of total lipids from microalgae, cyanobacteria, and wild mixed-cultures. Bioresour Technol 102(3):2724–2730. doi:10.1016/j.biortech.2010.11.026

    Article  CAS  Google Scholar 

  • Wang ZT, Ullrich N, Joo S, Waffenschmidt S, Goodenough U (2009) Algal lipid bodies: stress induction, purification, and biochemical characterization in wild-type and starchless Chlamydomonas reinhardtii. Eukaryot Cell 8(12):1856–1868. doi:10.1128/EC.00272-09

    Article  CAS  Google Scholar 

  • Wang Y, He B, Sun Z, Chen Y (2016) Chemically enhanced lipid production from microalgae under low sub-optimal temperature. Algal Res 16:20–27. doi:10.1016/j.algal.2016.02.022

    Article  Google Scholar 

  • Widjaja A, Chein CC, Ju YH (2009) Study of increasing lipid production from fresh water microalgae Chlorella vulgaris. J Taiwan Inst Chem E 40:13–20. doi:10.1016/j.jtice.2008.07.007

    Article  CAS  Google Scholar 

  • Wirshing A, Minocha S (2012) Genetic engineering of Dunaliella: potential for improved biofuel production. Honors Theses, Paper 23, Department of Biology, University of New Hampshire, Durham

  • Wong DM, Franz AK (2013) A comparison of lipid storage in Phaeodactylum tricornutum and Tetraselmis suecia using laser scanning confocal microscopy. J Microbiol Methods 95:122–128. doi:10.1016/j.mimet.2013.07.026

    Article  CAS  Google Scholar 

  • Yamaberi K, Takagi M, Yoshida T (1998) Nitrogen depletion for intracellular triglyceride accumulation to enhance liquefaction yield of marine microalgal cells into a fuel oil. J Mar Biotechnol 6(1):44–48

    CAS  Google Scholar 

  • Yao S, Brandt A, Egsgaard H, Gjermansen C (2012) Neutral lipid accumulation at elevated temperature in conditional mutants of two microalgae species. Plant Physiol Bioch 61:71–79. doi:10.1016/j.plaphy.2012.09.007

    Article  CAS  Google Scholar 

  • Yu WL, Ansari W, Schoepp NG, Hannon MJ, Mayfield SP, Burkart MD (2011) Modifications of the metabolic pathways of lipid and triacylglycerol production in microalgae. Microb Cell Fact. doi:10.1186/1475-2859-10-91

    Google Scholar 

  • Zhu LD, Li ZH, Hiltunen E (2016) Strategies for lipid production improvement in microalgae as a biodiesel feedstock. BioMed Res Int 2016:1–8. doi:10.1155/2016/8792548

    CAS  Google Scholar 

Download references

Acknowledgements

The authors would like to gratefully thank the German Academic Exchange Service (DAAD) in collaboration with the Ministry of Higher Education (MoHE) of the Arab republic of Egypt in the German Egyptian Long-Term Scholarship program (GERLS). We also would like to thank Miss. Angelina Stifanelli and Miss. Sian Lant at Nottingham Trent University, Nottingham, UK; Mrs. Noha Mohamed at New Mexico State University, USA for their revising and grammar corrections.

Author information

Authors and Affiliations

  1. Department of Life Sciences and Chemistry, Jacobs University Bremen gGmbH, Campus Ring 1, 28759, Bremen, Germany

    Khaled N. M. Elsayed & Tatiana A. Kolesnikova

  2. Institute of Environmental Biology and Biotechnology, University of Applied Sciences, 28199, Bremen, Germany

    Anja Noke & Gerd Klöck

  3. Department of Botany and Microbiology, Faculty of Science, Beni-Suef University, Beni-Suef, 62511, Egypt

    Khaled N. M. Elsayed

Authors
  1. Khaled N. M. Elsayed
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tatiana A. Kolesnikova
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anja Noke
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gerd Klöck
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Khaled N. M. Elsayed.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 8059 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Elsayed, K.N.M., Kolesnikova, T.A., Noke, A. et al. Imaging the accumulated intracellular microalgal lipids as a response to temperature stress. 3 Biotech 7, 41 (2017). https://doi.org/10.1007/s13205-017-0677-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s13205-017-0677-x

Keywords

Search

Navigation